Feline vaccine compositions and method for preventing chlamydia infections or diseases using the same

ABSTRACT

This invention provides a feline vaccine composition comprising an immunogenically active component having inactivated mammalian chlamydial cells or antigens derived therefrom, in combination with an effective amount of an immunogencally suitable adjuvant; and a veterinary pharmaceutically acceptable carrier or diluent. The vaccine composition is useful to prevent chlamydia, e.g.  C. psittaci,  infections or diseases in felines, and may also be combined with other vaccine compositions or therapy. A process for producing  C. psittaci  suitable for use in the production of safe and effective chlamydia vaccines, and a method for preventing chlamydia infections or diseases in felines, are also provided.

[0001] This application is a continuation of application Ser. No.08/467,775, filed Jun. 6, 1995 (now U.S. Pat. No. 6,004,563); which wasa continuation of Ser. No. 08/065,741, filed May 20, 1993, which was adivisional of Ser. No. 07/610,229, filed Nov. 7, 1990 (now U.S. Pat. No.5,242,686, issued Sep. 7, 1993). Each of these prior applications ishereby incorporated herein by reference, in its entirety.

[0002] The present invention relates to vaccines for veterinary use. Inparticular, the invention relates to vaccines comprising a combinationof one or more immunologically active components, i.e. inactivatedChlamydia psittaci or antigens derived therefrom, for prevention andtreatment of chlamydia diseases in mammals, such as cats. The inventionalso relates to methods for immunizing and treating such animals withsuch vaccines.

BACKGROUND OF THE INVENTION

[0003] Feline Chlamydia psittaci is the etiologic agent for a commonconjunctual and respiratory disease of cats known as feline pneumonitis(FPn) (Baker, J. A. (1942) Science 96:475-476). This highly contagiousdisease is characterized by sneezing and coughing and is accompanied bymucopurulent ocular and nasal discharges (Baker, J. A. (1944) J. Exp.Med. 79:159-172). All age groups of cats are susceptible and althoughmortality is not great, infected kittens and older animals may becomeseverely debilitated. Furthermore, because of its extremecommunicability, FPn constitutes a major problem in pet hospitals,clinics and catteries, etc.

[0004] Feline Chlamydial infection, like chlamydial infections in otherspecies which are clinically manifested by ocular disease, is notrestricted to the conjunctival mucosa. For example, it has been shownthat chlamydia inoculation into pathogen-free kittens producedconjunctivitis and rhinitis as well as positive identification ofchlamydia in the superficial gastric mucosa (Gaillard, E. T. et al.(1984) Am. J. Vet. Res. 45:2314-2321). In another study, C. psittaci wasrecovered from a female cat with persistent genital tract infection,resulting from ocular rather than direct genital infection (Darougar, S.M. et al. (1977) pages 186-198 in D. Hobson and K. K. Holmes (Ed.)Nongonococcal Urethritis and Related Infections, American Society forMicrobiology, Washington, D.C.). Persistent genital tract infection byC. psittaci is believed to be a cause of reproductive failure incatteries, however the mechanism of such failure is unclear. The mode ofextraocular transmission and the contribution of extraocular infectionto the persistence and pathology of feline chlamydial disease isunknown.

[0005] Vaccination studies with modified-live compositions has producedconflicting results. Modified live chlamydial vaccines in general haveshown variable efficacy ranging from no protection (Cello, R. M. J., Am.Vet. Med. Assoc. 158:932-938, 1971) to partial protection (Shewen, P.E., et al., Can. J. Comp. Med. 44:244-251, 1980) to almost completeprotection (McKercher, D. G., Am. J. Vet. Res. 13:557-561, 1952; Mitzel,J. R., and A. Strating, A. J. Vet. Res. 38:1381-1363, 1977; Kolar, J. R.and T. A. Rude, Feline Practice 7:47-50, 1977 and Vet. Med. S. A. Clin.76:1171-1173, 1981; Wills, J. M. et al., Infec. Immun. 55:2653-2657,1987). However, in studies demonstrating vaccine efficacy, chlamydia wasisolated in conjunctival swabs from vaccinated cats 24 days postchallenge (Mitzel and Strating, 1977), 31 days post challenge (Kohlerand Rude, 1977), and as late as 68 days post challenge (Wills et al.,1987) with sporadic isolations occurring between 3 and 8 months postchallenge (Wills, J. M. Ph.D. thesis, University of Bristol, England,1986). In addition, Wills et al. (1978) have demonstrated that excretionof chlamydia from vaccinated cats was actually prolonged when comparedto the controls. No difference could be demonstrated between thevaccinates and controls in the amount of chlamydial shedding from theeyes or the transmission of the organism to the gastrointestinal andgenital tracts.

[0006] Similar studies with inactivated chlamydial preparations producedmixed results. In one feline study where the efficacy of a killedpreparation was evaluated, irradiated and crystal violet treated,purified yolk sac suspensions were described and used (McKercher, D. G.,Am. J. Vet. Res. 13: 557-561, 1952). The induced protection wascomparable to that of modified-live vaccines similarly purified, but wasinferior to a modified-live crude yolk sac preparation. On the hand,comparative challenge studies conducted with four inactivated vaccinepreparations and a commercial modified-live vaccine demonstrated thatthe inactivated preparations conferred virtually no protection againstchlamydial infection in felines (Shewen, P. E. et al., Can. J. Como.Med. 44: 244-251, 1980).

[0007] The egg yolk sac-propagated FPn used to prepare the knownmodified-live and inactivated preparations is also known to contain alarge amount of a toxin (Hamre, D. et al., J. Infect. Dis. 74:206-211(1944). Release of this toxin, lipopolysaccharide, or anotherunidentified antigen(s) onto the eye during FPn infection may contributesignificantly to the pathogenesis of the ocular disease caused by thisorganism.

[0008] Because of the documented inability of prior modified livepreparations to reduce shedding, and the probability that modified-livevaccinated infected animals could shed both the vaccine and fieldstrains, with the concomitant risk of recombination, reactivation, andcommunication of disease to surrounding uninfected animals, the needexists for the development of safe, efficacious inactivated C. psittacivaccines. Moreover, prior inactivated preparations have beenunsuccessful or markedly inferior to modified-live preparations.

OBJECT OF THE INVENTION

[0009] It is an object of the present invention to provide aninactivated feline chlamydia vaccine composition having high antigenload in combination with a potent adjuvant which will elicitcorresponding antisera when administered systemically to a subjectfeline.

[0010] It is another object of the present invention to provide aprocess for producing C. psittaci suitable for use in the production ofsafe, effective chlamydia vaccines.

[0011] It is a further object of the present invention to provide amethod for preventing chlamydia infection in felines by immunizing theseanimals with an efficacious inactivated vaccine composition.

[0012] These and other objects will become more apparent in light of thedetailed description which follows.

SUMMARY OF THE INVENTION

[0013] The present inventors have discovered that an immunogenicallyactive component can be made and usefully incorporated into a vaccinecomposition for preventing chlamydia infections in mammals, i.e.felines. The immunogenically active component has inactivated, e.g.,chemically inactivated, chlamydial (chlamydia) cells or antigens derivedtherefrom, such as outer membrane extracted antigens. Theimmunogenically active component is combined with an effective amount ofan adjuvant, and a veterinary pharmaceutically acceptable carrier ordiluent therefor.

[0014] The present invention also provides a purification processwhereby toxic immunogenic substances in egg yolk sac cultures containingC. psittaci are removed when said cultures are further subcultured inmammalian cells, i.e. dog kidney cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a plot of daily mean temperature response versus dayspost challenge of two groups of non-vaccinated felines challenged withtwo low egg passage chlamydia preparations.

[0016]FIG. 2 is a plot of the daily mean clinical sign score versus dayspost challenge of two groups of non-vaccinated felines challenged as inFIG. 1.

[0017]FIG. 3 is a plot of the mean clinical sign score versus days postchallenge of five vaccinated and one unvaccinated group of felineschallenged with a combination of the two low egg passage chlamydiapreparations. The inactivated chlamydia vaccine compositions containedSaponin/AlPO₄ adjuvants.

[0018]FIG. 4 is a combined plot of the mean clinical sign scores andtemperature response versus days post challenge of five vaccinated andone unvaccinated group of felines challenged as in FIG. 3. Theinactivated chlamydia vaccine compositions contained Saponin/AlPO₄adjuvants.

[0019]FIG. 5 is a combined plot of the mean clinical sign scores andfever response versus days post challenge of two groups of vaccinatedand one unvaccinated group of felines challenged with a low egg passagechlamydia isolate. The inactivated chlamydia vaccine compositionscontained EMA/Neocryl®/MVP adjuvants.

DETAILED DESCRIPTION OF THE INVENTION

[0020] All literature references, patents and patent applications citedin this specification are hereby incorporated by reference in theirentirety.

[0021] The present invention provides a vaccine composition comprisingan immunogenically active component having inactivated Chlamydiapsittaci or antigens derived therefrom in combination with an effectiveamount of an adjuvant; and a veterinary pharmaceutically acceptablecarrier or diluent therefor.

[0022] As used herein, the term “immunogenically active” componentrefers to the ability of the component described herein to stimulate animmune response, i.e., to cause the production of antibodies and/or acell-mediated response when introduced into a subject (mammal, e.g.feline). More specifically, the term “immunogenically active” componentrefers to the ability of this component to stimulate secretory antibodyand/or cell-mediated response production in local mucosal regions, e.g.the surface of the upper respiratory tract and/or conjunctiva, and thecervico-vaginal cavity, when administered systemically as a vaccinecomposition according to the present invention.

[0023]Chlamydia psittaci organisms used to make the immunogenicallyactive components of the present vaccine composition can be obtainedfrom university or research institutes, e.g. National VeterinaryServices Laboratory (NVSL) (Ames, Iowa) or can be isolated from thefluids or tissues of infected mammals. Such sources include, forexample, blood, vaginal, cervical, ocular, pneumonic, gastral, nasalfluids, discharges, secretions and scrapings. In particular, the viralpathogens can be isolated from ocular and nasal secretions. IsolatedChlamydia psittaci can best be maintained in egg yolk cultures.

[0024] According to the invention, a chlamydia isolate obtained fromNVSL (Lot No. 87-16-1) was first propagated in an egg yolk culture. Seedchlamydia from this isolate was diluted in Dulbecco's phosphate bufferedsaline (PBS), pH 7.0, and inoculated into the egg yolk sac of six orseven day old embryonated SPF eggs at the concentration of 100 to 1000ELD₅₀ per egg. The diluted seed was provided in a volume of 0.1 ml. Theeggs were wiped with a 2% tincture of iodine mixture or similar chemicaldisinfectant. A small hole was drilled in the middle of the air sac andthe inoculum inserted into the yolk sac with a 22 gauge needle. The holewas sealed with glue. The inoculated embryonated eggs were incubated forfive to ten days in 37-C±1° C. incubator. The eggs showed normaldevelopment and growth until day 6 or 7 post inoculation, when thevascular system starts to break down, indicating embryo death fromchlamydia. Contamination would be observed by the death of the embryoduring the first three days after inoculation. All eggs dying up to day3 and other eggs demonstrating signs of contamination were discarded.Elapsed time between inoculation and harvest was five to ten days forthe embryonated eggs. The eggs were kept at 2-9° C. for two to fourhours before harvest.

[0025] A usual intermediate seed harvest consisted of approximately 200infected embryonated eggs. The egg yolk and chorioallantoic membranesfrom up to 100 eggs were combined into sterile pooling containers foreach lot. Up to a 50% volume of PBS was added to the containers and theinfected membranes were blended in a mechanical blender. The infectedmixture was centrifuged at 1000 rpm for 15 minutes and the PBS middlelayer containing chlamydial elementary bodies was drawn off. A stock of40% sorbitol was added (to a final concentration of 10%) as astabilizer. Other stabilizers, such as sucrose, NZ Amine, and SGGK mayalso be used.

[0026] The harvested material was aliquoted into 100 ml amounts andfrozen at −50° C. until used as seed for at least one subsequent passagein a suitable cell line. For example, chlamydia can be subcultured incell lines derived from sources such as feline, canine, avian, equine,ovine, caprine, bovine, swine, mouse and human, said cell linescomprising fibroblasts or epithelial, synovium, lung, spleen, amnion,stomach, kidney, cornea, liver, testicles, laryngeal tumor and HeLacells. Dog kidney cells are preferred as a cell line for subculturingyolk-sac propagated chlamydia.

[0027] A dog kidney cell line [MDCK(NBL-2)] was purchased from theAmerican Type Culture Collection (ATCC No. CCL-34, passage no. 55),subcultured, and stored at −70° C. or lower before use. The dog kidneycell growth medium consisted of Eagle's minimum essential media (MEM) towhich the following may be added: not more than 10% bovine serum orserum substitutes, not more than 0.5% lactalbumin hydrolysate, not morethan 0.5% bovine serum albumin, Neomycin at a concentration of 30mcg/mL, and not more than 2.5 mcg/mL Amphotericin B. Kidney cellmonolayers can be grown in 850 or 1750 cm² disposable roller bottles,1700 or 3500 cm² disposable pleated roller bottles or in bioreactorscapable of holding 10 to 2000 L volumes.

[0028] Just prior to tissue culture inoculation, seed chlamydia wasdiluted in a chlamydia inoculation medium which consisted of Eagle's MEMwith the following additions: not more than 0.5% lactalbuminhydrolysate, not more than 10 mcg/mL polybrene, Neomycin at aconcentration of 30 meg/mL and not more than 2.5 mcg/mL Amphotericin B.Growth medium was discarded when cell sheets were approximately 100%confluent. The diluted inoculum was added aseptically to productioncontainers at a MOI of 1:10 to 1:1000 for tissue culture. The inoculumwas adsorbed for up to 24 hours at 37° C. At the end of the adsorptionperiod, the inoculum was discarded, the monolayer was rinsed andchlamydia propagation medium was added.

[0029] Chlamydia propagation medium consisted of Eagle's MEM with thefollowing additions: not more than 0.5% bovine serum albumin, and/or0.5% bovine serum and/or 0.5% lactalbumin hydrolysate, not more than0.0158 M sucrose, not more than 20 mM Hepes, Neomycin at a concentrationof 30 mcg/mL, and not more than 2.5 mcg/mL Amphotericin B. Chlamydiapropagation media was added to production flasks as follows: up to 500mL for 850 cm² disposable roller bottles, up to 1000 mL for 1750 cm²disposable roller bottles and 1700 cm² disposable pleated rollerbottles, and up to 2000 mL for 3500 cm² disposable pleated rollerbottles. Chlamydia propagation media could also be added to bioreactorvessels, if used, as follows: up to 10 L for 10 L vessels, up to 30 Lfor 30 L vessels, up to 100 L for 100 L vessels, up to 400 L for 400 Lvessels and up to 2000 L for 2000 L vessels.

[0030] After inoculation, the cells do not exhibit cytopathology.Contamination would be observed by clouding of the medium, and anyunsatisfactory or questionable cultures are eliminated. Tissue cultureswere incubated for 7 to 21 days at 33-37° C. Tissue culture vessels maybe examined for indications of adequate chlamydial growth by making acell smear from the infected monolayer of one of the tissue culturevessels and checking the smear by indirect immunofluorescence assay(IFA) for characteristic inclusion bodies. Vessels were also inspectedfor gross indications of pH changes and contamination.

[0031] Elapsed time between inoculation and harvest was 7-21 days forthe tissue culture passages. Only cultures considered free of bacteriaand fungi by macroscopic examinations were harvested.

[0032] For a single harvest, fluids were harvested aseptically byremoving the contents of the culture bottles into sterile poolingcontainers. To remove the infected cell monolayers, MEM containing1:20,000 thimerosal at a volume of up to one-tenth the chlamydialpropagation media was added to each vessel. The vessel was rolled at 37°C. until the infected monolayer started to come off (up to 4 hours). Themedia containing the infected cells was combined with the supernatantsand enough thimerosal was added to the total volume to bring thethimerosal concentrations to 1:20,000. The thimerosal serves the dualpurpose of removing the infected monolayer from the bottle andinactivating the live chlamydia. Alternatively, the infected monolayerof cells may be removed by freezing the bottles at −20° C. or below for4 to 24 hours. Samples may be taken from the pooled supernatants plusinfected cells for determination of the Enzyme Linked ImmunosorbentAssay (ELISA) antigenic value and for sterility and inactivation tests.

[0033] For multiple harvest of infected materials, the cultures may berefed and harvested up to six times by adding chlamydia propagationmedia to the bottles or bioreactor and incubating for an additional 5 to14 days for each multiple harvest. The final harvest shall consist ofsupernatants and infected cells harvested by the above-describedprocedures. Fluids for vaccine use may consist of supernatant onlyand/or supernatants plus infected cells.

[0034] As one measure of vaccine potency, each individual or pooled lotshould have an acceptable ELISA antigen value as measured against areference vaccine. Inactivated chlamydia may be concentrated and/orpooled with other harvests, such that the averaged antigenic value meetsor exceeds the minimum acceptable value.

[0035] Inactivated chlamydial fluids may be concentrated up to 20 fold,if necessary, by ultrafiltration, with a molecular weight cutoff of100,000 daltons, or by differential centrifugation. Concentrates werestored at 2-9° C. until mixed or microfluidizied with adjuvant.

[0036] Following their harvest and separation as whole cell isolates,chlamydia may be inactivated by conventional inactivation means. Forexample, inactivation of whole cell isolates can be achieved bycontacting the cells with an inactivating agent. Suitable agentsinclude, without limitation, binary ethylenimine, betapropiolactone,formalin, merthiolate, thimerosal, gluteraldehyde, sodium dodecylsulfate, triton-100, acetone, ether, phenol, heat (e.g. 56° C. for 5 ormore minutes), ultraviolet irradiation in the presence or absence ofpsoralen, gamma irradiation, or a combination of any of these agents inan aqueous suspension. Preferred as a chemical inactivating agent isthimerosal at a final concentration of 1:20,000 for 3 days.

[0037] After inactivation, the inactivated chlamydial (chlamydia) wholecells can be adjusted to an appropriate concentration which meets orexceeds the minimum acceptable ELISA antigen value in combination withan immunogenically stimulating adjuvant. The preinactivation chlamydiatiters of such antigen preparations generally range from 10^(5.0) to10^(6.5) ELD₅₀ per dose. When antigens derived from chlamydial(chlamydia) cells, e.g., C. psittaci, are employed, a suitable amount ofprotein or antigen per dose may be used, for example, 50 to 1,000ug/dose.

[0038] As used herein the term “immunogenically stimulating adjuvant”refers to an agent, compound or the like, which potentiates orstimulates the immune response in a subject animal when administered incombination with the inactivated whole cells. Thus, the immune response,elicited by the inactivated whole cell-adjuvant combination, as measuredby antibody and/or cell-mediated response, will generally be greaterthan that provoked by the inactivated whole cells alone.

[0039] The immunogenically stimulating adjuvants augment the immuneresponse provoked by the inactivated chlamydia cells. The inactivatedchlamydia cells may or may not elicit a desired immune response, e.g., alocal mucosal and/or a strong systemic immunity, when systemicallyadministered alone. An essential feature of the present invention is thecombination of the inactivated chlamydia cells and immunogenicallystimulating adjuvant, which provide the desired immune response.

[0040] Non-limiting examples of the immunogenically stimulatingadjuvants used in the practice of the present invention are surfactants,e.g., hexadecylamine, octadecylamine, lysolecithin,dimethyldioctadecyl-ammonium bromide, N,N-dioctadecyl-N′-N-bis(2-hydroxyethylpropane diamine), methoxyhexa-decylglycerol and pluronicpolyols, saponin, Quil A; polyanions or polycations, e.g., pyran,diethylaminoethyl (DEAE) dextran, dextran sulfate, polybrene, poly IC(polynucleotide complex of polyinosinic-polycytidylic acid) polyacrylicacid, carbopol, aluminum hydroxide, aluminum phosphate; peptides, e.g.,muramyl dipeptide, dimethylglycine, tuftsin; oil emulsions,immunomodulators, e.g., interleukin-1, interleukin-2; interferon(s); orcombinations of any of the foregoing adjuvant agents.

[0041] A number of acrylic acid polymers and copolymers of acrylic acidand methacrylic acid and styrene have adjuvant activity. PolyvinylChemical Industries (Wilmington, Mass.) provide such polymers under thetradename NEOCRYL®. NEOCRYL A640, an aqueous acrylic copolymer having pH7.5, viscosity 100 eps (Brookfield 25° C.), a weight per gallon of 8.6pounds as supplied containing 40% solids by weight, 38% solids by volumeand an acid number of 48, is a preferred adjuvant. Specifically, NEOCRYLA640 is an uncoalesced aqueous acrylic copolymer with styrene. Morespecifically, NEOCRYL A640 is a latex emulsion of a copolymer of styrenewith a mixture of acrylic and methacrylic acid. Other useful NEOCRYLproducts are 520 and 625, and NEOREZ 966.

[0042] Ethylene/maleic anhydride copolymer is another preferredadjuvant. Suitable ethylene/maleic anhydride copolymers useful in thisinvention are the linear ethylene/maleic copolymers such as EMA-31 (asproduced by Monsanto Co., St. Louis, Mo.), a copolymer withapproximately equal amounts of ethylene and maleic anhydride, having anestimated average molecular weight of about 75,000 to 100,000. Thesecopolymers are water soluble, white, free-flowing powders having thefollowing typical properties: a true density of about 1.54 g/mL, asoftening point of about 170° C., a melting point of about 235° C., adecomposition temperature of about 274° C., a bulk density of about 20lbs/ft³, and a pH (1% solution) of 2.3.

[0043] More preferably, two or more adjuvants will be admixed with theharvested inactivated chlamydiosis (chlamydia) cells or antigens derivedtherefrom. One preferred combination is ethylene/maleic anhydridecopolymer, NEOCRYL A640, and MVP. MVP is a mineral oil adjuvant producedby Modern Veterinary Products (Omaha, Nebr.). Also preferred as animmunogenically stimulating adjuvant is a combination of saponin andaluminum phosphate.

[0044] It has been discovered that the adjuvants described above willact in effective amounts to immunogenically stimulate the inactivatedchlamydia cells or antigens derived therefrom. As used herein, theeffective amount of the immunogenically stimulating adjuvant cancomprise from about 0.01% to about 50%, preferably from about 1% toabout 5% for EMA/Neocryl®/MVP, and preferably from about 0.01% to about0.3% for saponin/aluminum phosphate.

[0045] The vaccine composition of the present invention also comprises aveterinary pharmaceutically acceptable carrier or diluent. A preferredcarrier is saline.

[0046] As further embodiments of the present invention, the vaccinecomposition can be administered, for example, by incorporating theactive component into liposomes. Liposome technology is well-known inthe art having been described by Allison, A. C. and Gregoriades, G.,Liposomes as Immunologic Adjuvants, Nature 252:252054 (1974) and Dancy,G. F., Yasuda, T., and Kinsky, S. C., J. Immunol. 120:1109-13 (1978). Inaddition, the active component can be conjugated to suitable biologicalcompounds or materials, such as, for example, polysaccharides, peptides,proteins, or a combination of any of the foregoing. Conjugated vaccinesare described by Coon, J., and Hunter, R. L., J. Immunol. 110:183-90(1973).

[0047] Also as further embodiments of the present invention, the vaccinecomposition can be administered as sustained release product(s), forexample, by incorporating the vaccine components in polymers, e.g.lactide-glycolide copolymer. Microparticles were previously found topossess adjuvant effect for an entrapped antigen following parenteraladministration, as described by Sjoholm, I. and Edman, P., Microsoheresand Drug Therapy in Pharmaceutical, Immunological and Medical Aspects(Editors Davis, S. S., Illum, I., McVie, J. G. and Tomlinson, E.)Elsevier, Amsterdam, 1984, P. 245-262.

[0048] It is advantageous to formulate the vaccine composition of thisinvention in a dosage unit form to facilitate administration and insureuniformity. Thus, in another embodiment, this vaccine composition can beformulated in dosage unit form comprising at least about 2×10⁴inactivated chlamydia cells, preferably at least about 1×10⁵ cells.

[0049] In a further embodiment, the vaccine composition can comprise aparenteral injectable form, again to ease its administration to asubject feline.

[0050] The present invention provides a method for preventing chlamydiainfection in felines comprising administering to a feline an effectiveamount of the vaccine composition described above.

[0051] The routes of administration contemplated by the presentinvention are parenteral, e.g., subcutaneous, intramuscular,intraperitoneal and intradermal. Preferred routes of administration aresubcutaneous and intramuscular.

[0052] It has been discovered that the vaccine composition of thepresent invention is useful to prevent chlamydia infection in felinesthat need such protection when administered parenterally, e.g.,subcutaneously or intramuscularly, in effective amounts. An effectiveregimen of treatment includes administering the vaccine composition, forexample, in dosage unit form as described above, at least about twotimes, with each administration separated by about two (2) to about four(4) weeks, preferably from about fourteen (14) to about thirty (30) daysor so.

[0053] It will be understood by the skilled practitioner that thevaccines of the present invention may be combined with other vaccines toproduce a combination vaccine effective against more than one pathogen.Examples include a chlamydia vaccine in combination with one or morevaccines for feline leukemia, panleukopenia, calici, rhinotracheitis,feline acquired immunodeficiency disease, rabies, feline infectiousperitonitis, toxoplasmosis, and Borrelia burgdorferi.

[0054] The working examples set forth below are intended to illustratethe invention without limiting its scope.

EXAMPLE 1

[0055] Vaccine Preparations

[0056] Killed feline chlamydia psittaci (FCP) vaccines were formulatedto contain thimerosal-inactivated C. psittaci in combination with killedfeline leukemia (FeLV), panleukopenia (FPV), calici (FCV) andrhinotracheitis (PCT) viruses. The vaccines contained eitherAlPO₄/saponin or EMA/Neocryl/MVP as adjuvants. As shown in Table 1, sixformulations were made, and these were compared with a commercialmodified-live product. TABLE 1 Vaccine Adjuvant FCP^(a) Ratio FCP-ELISAFCP 1A Saponin/AlPO₄ 1   1.00^(b) FCP 1B Saponin/AlPO₄ 1/5 0.20 FCP 2ASaponin/AlPO₄ 1   0.81 FCP 2B Saponin/AlPO₄ 1/5 0.11 FCP 3AEMV/Neocryl ®/MVP 1   1.00^(c) FCP 3B EMV/Neocryl ®/MVP 1/5 0.36Eclipse ®4 N.D d N.D

[0057] a. This ratio represents the amount of the FCP component in thecombination vaccine composition comprising FCP, FeLV, FVR, FCV and FPVcomponents.

[0058] b. FCP 1A served as a reference vaccine for the FCP 1B and FCP 2AELISA potency determinations, with a value of 1.00.

[0059] c. FCP 3A served as a reference vaccine for the FCP 3B ELISApotency determination, with a value of 1.00.

[0060] d. The Eclipse®4 modified-live commercial combination vaccine hadmodified-live chlamydia as the immunogenic component and is commerciallyavailable from Solvay Animal Health, Inc. Because the adjuvant systemfor Eclipse®4 is unknown, a meaningful relative FCP-ELISA potency valuecannot be determined.

EXAMPLE 2

[0061] Challenge and Isolation of C. psittaci in Felines

[0062] Two challenge preparations were evaluated in young cats in orderto produce consistent disease due to infection with Chlamydia psittaci.

[0063] A. Experimental Animals

[0064] Ten animals used for this experiment were specific pathogen free(SPF) cats purchased from Liberty Laboratories (Liberty Corner, N.J.).The cats were screened after receipt of antibodies to C. psittaciutilizing an ELISA assay for C. psittaci antibodies. The SPF cats were10 to 12 weeks of age at the time of vaccination and approximately 16 to18 weeks old at the time of challenge.

[0065] B. Experimental Design

[0066] Two 1 mL vaccinations of the FCP 1 and FCP 2 vaccines orfractional dose vaccines were administered intramuscularly 21 daysapart. Two doses of Solvay's modified-live Eclipse®4 vaccine were given21 days apart according to the label directions. Animals receiving themodified live vaccine were held in separate facilities both before andafter vaccination to prevent any possible spread of the vaccine strainto other cats. All cats were challenged 22 days post second vaccinationand were bled at the time of the first and second vaccinations, and thenweekly until at 28 days post challenge.

[0067] C. Challenge Preparations

[0068] The first challenge preparation was a feline pneumonitis isolate(Chlamydia psittaci, strain Cello) which represents the 6th egg yolk sacpassage of the material described by Dr. Robert Cello (Am. J. Opth.63:1270-1273, 1967). This reagent (Lot # 87-16-1, National VeterinaryServices Laboratory) is a 20% yolk sac suspension in brain heartinfusion broth and was stored at −70° C. until use. This challengeisolate was further passaged once in egg yolk to produce a secondchallenge preparation (i.e. low egg passage challenge reagent). Thechallenge evaluation indicated that both materials can adequately infectcats and produce chlamydial disease (FIGS. 1 and 2).

[0069] D. Challenge Procedure

[0070] Two groups of five 14 week old SPF cats were challenged witheither the NVSL feline pneumonitis challenge material or with the lowegg passage FCP preparation derived from the NVSL challenge material.The total amount of Chlamydia given to each cat was 10^(5.5) ELD₅₀ forthe NVSL challenge group and 10^(6.375) ELD₅₀ for the low egg passagedchallenge group. The challenge material was nebulized into the eyes andnares of each cat over a period of 2-3 minutes. Rectal temperatures,conjunctival swabs, throat swabs and blood for serum were taken atintervals for 53 days. The animals were observed daily for clinicalsigns for 28 days.

[0071] E. Chlamydia Detection and Post-Challenge Evaluation

[0072] 1. Chlamydia Detection

[0073] For the challenge evaluation, conjunctival smears were made fromboth eyes of each cat at intervals starting from the day of challengeuntil the end of the study. Smears were fixed in acetone, stained bydirect FA using FITC-conjugated mouse monoclonal antibody directedtoward the chlamydial group antigen (Bartels Immunodiagnostic Supplies,Inc.) and examined for the presence of Chlamydia in ocular cells.

[0074] 2. Post-Challenge Evaluation

[0075] All cats in the challenge evaluation were monitored for 24 hoursprior to challenge to establish baselines and for 28 days followingchallenge. Rectal temperatures and gross clinical signs, including butnot limited to ocular discharge, nasal discharge, coughing, sneezing,conjunctivitis, anorexia and depression were recorded daily.Conjunctival smears were taken at intervals starting the day ofchallenge. Cats were bled weekly starting from the day before challengeand at 7, 14, 21, and 28 days post challenge.

[0076] The following criteria were measured and recorded:

[0077] Temperature Response

[0078] Temperature scoring was based on the following scale: ° F. Score<103.0 0 103.0-103.9 1 104.0-104.9 2 ≧105.0 3

[0079] Gross Clinical Signs

[0080] Clinical points were assigned according to the following scale:Clinical Sign Score Ocular discharge Clear (Serous) mild 0.5 moderate1.0 Mucopurulent mild 1.0 moderate 1.5 severe 2.0 Nasal discharge Clear(Serous) mild 0.5 moderate 1.0 Mucopurulent mild 1.0 severe 2.0 Sneezing1.0 Coughing 1.0 Anorexia 1.0 Depression 1.0 Dehydration 1.0 Dyspnea 2.0Conjunctivitis mild 0.5 moderate 1.0 severe 2.0

[0081] Statistical analysis of the temperature response data wasaccomplished using the Student t-test. Clinical sign data was analyzedby the Student t-test and/or the Mann-Whitney Rank Test. These analyseswere performed using the software package Statview 512+ from BrainPower, Inc. on an Apple MacIntosh SE computer.

[0082] F. Challenge Results

[0083] 1. Temperature Response in Cats Following Challenge

[0084] The daily mean temperature response in both challenge groups isshown in FIG. 1. A consistent elevation in temperature is evident inboth groups starting from day 9 in the NVSL challenge group and day 12in the low egg passage group. Temperatures in both groups remainedelevated until 25-26 days post challenge, then returned to normal (<103°F.). The fever response peaked at 13 days following challenge for bothgroups. The peak of the average fever response was 105° F. for catsreceiving the NVSL challenge preparation and 105.3° F. for thosereceiving the low egg passage preparation. These data indicate that thetemperature response for each challenge material paralleled the otherexcept for a lightly slower initial elevation with the low egg passagechallenge material.

[0085] 2. Clinical Sign Scores of Cats Following Challenge

[0086]FIG. 2 shows the, daily mean clinical score for both challengegroups. Peak clinical signs occurred between days 7 through 13 and days17 through 22 with both challenge preparations, although the relativelevel of clinical signs stimulated by the low egg passage preparation inthe first peak was lower than by the NVSL challenge preparation.

[0087] 3. Detection of Chlamydia in Cats Following Challenge

[0088] Chlamydia shedding occurred in all cats following challenge.Chlamydia was demonstrated in conjunctival smears starting at 5 dayspost challenge. Chlamydial isolations were more sporadic in the low eggpassage challenge group initially than in the NVSL challenge group.Conjunctival smears in all of the cats were positive on day 25 postchallenge.

EXAMPLE 3

[0089] Correlation of Immunogenicity and Potency of FCP VaccinesContaining Saponin/AlPO₄ as Adjuvants

[0090] A total of 91 SPF cats, 10 to 12 weeks of age, were utilized inthis study. The FCP 1A vaccination group consisted of 21 cats. The FCP1B and FCP 2A vaccination groups consisted of 20 cats each. The FCP 2Band the Solvay Eclipse®4 vaccination groups included 11 cats each. Thenon-vaccinated control group consisted of 11 cats. The reagent used tochallenge the cats in the immunogenicity trials was a combination of theNVSL challenge preparation and the low egg passage preparation dilutedto 10^(5.79) FLD₅₀ titer per cat. Results indicate a reliable andaccurate reproduction of disease from the use of the combined low eggpassage preparation and NVSL challenge preparation.

[0091] A. Reduction in Fever in Vaccinated Cats as Compared to ControlsFollowing Challenge

[0092] The temperature response of each animal in the five vaccine andcontrol groups were measured. The peak in the average temperatureresponses observed are summarized below: Days Post- Mean % Days VaccineGroup Challenge Temperature of Fever FCP 1A 20 102.6° F. 10.6 FCP 1B3-20 102.5° F. 96 FCP 2A 10 102.8° F. 17.6 FCP 2B 9-11 102.9° F. 20.1Eclipse ®4  9 102.7° F. 12.8 Controls  9 104.3 34.3

[0093] These results indicate that the two full strength FCP 1A and FCP2A vaccines of the invention and the FCP 1B fractional dose vaccineprovided significant differences in the temperature response versus thenon-vaccinated controls.

[0094] Furthermore, there was a significant reduction in the averagetemperature response varying from five to nine out of the 10 days duringwhich the mean temperature of the controls were equal to or greater than103.0° F. (indicating fever). These observations are summarized below:Total Days Showing Significant In Vitro Vaccine Group Difference fromControls Potency Value FCP 1A 9 1.0  FCP 1B 8 0.2  FCP 2A 8 0.81 FCP 2B4 0.11 Eclipse ®4 5 N.D.

[0095] These results indicate a positive correlation between ELISAantigen values (relative to FCP 1A, Example 1) and efficacy in reducingtemperature response in the vaccinates following challenge. The FCP 1A,1B, and FCP 2A vaccines were effective in reducing temperature response,whereas FCP 2B, with an ELISA value of 0.11, was not effective. The FCP1A, 1B and 2A vaccines were more efficacious than the modified-livevaccine with respect to protection against fever induced by chlamydiachallenge.

[0096] B. Reduction in Clinical Sign Scores in Vaccinated Cats asCompared to Controls Following Challenge

[0097] The mean clinical sign scores per day per group were calculatedaccording to the actual number of observation days, and are summarizedgraphically in FIG. 3. There was a significant reduction (p <0.05) inclinical sign scores in each vaccine group, varying from 16 to 24 out ofthe 24 days during which the non-vaccinated controls had clinical signscores greater than zero. These observations can be summarized asfollows: Total Days Showing Significant In Vitro Vaccine GroupDifference from Controls Potency Value FCP 1A 22 1.0 FCP 1B 23 0.2 FCP2A 24 0.81 FCP 2B 16 0.11 Eclipse ®4 22 NT

[0098] The least protected group was the FCP 2B fractional dose group,although it provided statistically significant reduction in clinicalsigns versus the control group. In addition, there was no statisticaldifference in the reduction in clinical signs afforded by the FCP 1A,1B, 2A and 2B vaccines compared to the modified-live vaccine.

[0099] The percent reduction in the mean clinical scores when comparedto the mean of the controls was 76.4% for FCP 1A, 80.1% for FCP 1B,81.1% for FCP 2B, 56.7% for FCP 2B and 79.3% for the modified-liveproduct. Significant differences between each vaccine group and thecontrol group were found. This confirms that the inactivated chlamydialvaccines of the invention are as or more efficacious than themodified-live vaccine.

[0100] C. Reduction in Combined Clinical Sign and Temperature Scores inVaccinated Cats as Compared to Controls Following Challenge

[0101]FIG. 4 combines the total clinical sign scores with the scoringfrom the temperature responses and are in general agreement with thedata scored separately. Significant lower combined scores (p<0.05) werefound in each vaccine group, varying from 21 to 24 out of the 24 daysduring which the non-vaccinated controls had clinical sign scoresgreater than zero. These observations are again summarized below: TotalDays Showing Significant Vaccine Group Difference from Controls FCP 1A23 FCP 1B 24 FCP 2A 24 FCP 2B 22 Eclipse ®4 23

[0102] The percent reductions in combined scores when compared to themean of the controls was 77.8% for FCP 1A, 80.5% for FCP 1B, 75.7% forFCP 2A, 53.6% for FCP 2B and 77.1% for Solvay Eclipse®4. As seen for theseparate clinical sign score data, significant differences between eachvaccine group and the control group were found.

[0103] In conclusion, all non-vaccinated control cats developedChlamydia disease following FCP challenge. The FCP 1A and FCP 2Avaccines and even their corresponding fractional dose vaccines (1B and2B) were efficacious in protecting vaccinated cats against virulentfeline C. psittaci challenge. These vaccines and their fractional doseswere effective in reducing both fever response and clinical signs in thevaccinates following FCP challenge. Moreover, the inactivated Chlamydiavaccines of the invention were found to be as or more efficacious as theknown modified life vaccine.

EXAMPLE 4

[0104] Post Challenge Evaluation of Felines Vaccinated with FCPCompositions Containing EMA/NEOCRYL®/MVP Adjuvant System

[0105] The efficacy of FCP vaccines containing EMA/Neocryl®/MVP asadjuvants was evaluated in young cats via vaccination and challengestudies. The vaccines were administered intramuscularly orsubcutaneously in two doses, 21 days apart. Efficacy of the FCP vaccineswas demonstrated by challenging the vaccinated groups and age-matchcontrols with the low egg passage virulent pneumonitis preparation asdescribed below.

[0106] A total of 100 healthy, specific pathogen free (SPF) cats werepurchased for the FCP vaccine efficacy testing. Ninety-five (95) catswere purchased from Liberty Laboratories (Liberty Crossing, N.J.) andfive (5) cats were acquired from Harlan-Spraque Dawley (Indianapolis,Ind.). All test animals were approximately 16 to 20 weeks of age at thetime of vaccination and approximately 20 to 25 weeks of age at the timeof challenge. These animals were housed in isolation facilitiesthroughout the entire study.

[0107] A total of 35 animals were divided into two vaccination groupsand one non-vaccinated control group. The 20 cats in the firstvaccination group, receiving 1 mL of the full dose of the FCP 3A vaccine(Example 1), were further subdivided into two groups with 10 catsreceiving the vaccine subcutaneously and 10 cats receiving the vaccineintramuscularly. A second dose of the vaccine was given 21 days later.Each of the five cats in the second vaccination group received 1 mL ofthe ⅕th fractional dose FCP 3B vaccine intramuscularly. A second dose ofvaccine was given 21 days later. The remaining 10 cats served asnon-vaccinated controls.

[0108] Twenty one (21) days following the second vaccination, all testcats were challenged with the aforementioned virulent feline pneumonitispreparation. The challenge material was prepared as the first egg yolkpassage material from a stock provided by NVSL. The material wasaliquoted and stored at −100° C. until use. At the time of challenge,this material was thawed and diluted 1:5 with PBS, to contain 10^(5.375)ELD₅₀. The Chlamydia was nebulized into the eyes and nares of the catsover a period of two to three minutes. Rectal temperatures and clinicalsigns were taken daily for 28 days. Conjunctival swabs and blood forserum were taken at weekly intervals.

[0109] The following criteria were measured and recorded according tothe scoring system described below: Fever/Clinical Sign Score Feverresponse <103.0° C. 0 103.0-103.9° C. 1.0 104.0-104.9° C. 2.0 ≧105.0° C.3.0 Ocular discharge Clear (serous) mild 0.5 moderate 1.0 Mucopurulentmild 1.0 moderate 1.5 severe 2.0 Nasal discharge Clear (serous) mild 0.5moderate 1.0 Mucopurulent mild 1.0 moderate 1.5 severe 2.0 Sneezing 1.0Coughing 1.0 Anorexia 1.0 Depression 1.0 Dehydration 1.0 Dyspnea 2.0Death 5.0 Conjunctivitis mild 0.5 moderate 1.0 severe 2.0

[0110] Analysis of the post-challenge fever and gross clinical signs(i.e. combined fever/clinical sign score) was accomplished by theMann-Whitney Rank Test. All analyses were performed using the softwarepackage Statview 512+ from Brain Power, Inc. on an Apple Macintosh II orSE computer.

[0111] The FCP 3A and 3B vaccines demonstrated satisfactory efficacyagainst the Chlamydia challenge. FIG. 5 shows the mean fever responseand clinical sign scores of vaccinated and control cats following FCPchallenge.

[0112] As expected, the virulent FCP challenge produced a significantly(p<0.05) higher fever response and chlamydial clinical signs in thenon-vaccinated controls, as compared to the vaccinates (FIG. 5).Following an incubation period of approximately 7±3 days, the virulentFCP-induced fever and clinical signs became increasingly apparent in thecontrol animals and peaked at day 19 post challenge with a combinedfever and clinical sign score of 4.35 per animal. Throughout the 28 daypost-challenge observation period, the average daily fever/clinical signscore for the controls was 2.19 per animal (FIG. 5).

[0113] Comparatively, the degree of the post-challenge fever/clinicalsign score for the vaccinates was much milder than that of the controls(FIG. 5). The average daily fever/clinical sign score for the FCP 3Avaccine group and the FCP 3B vaccine group were 1.34 and 1.23 peranimal, respectively, as demonstrated in FIG. 5. In addition, during theperiod when chlamydial disease was overt in the control group (i.e. day12 through day 28 post challenge), the fever/clinical sign score ofeither vaccine group was consistently less than that of the controlgroup. Statistically, there is a significant difference (p<0.05) whenthe average daily fever/clinical sign score of either vaccine group wascompared to that of the controls. Furthermore, when the dailyfever/clinical sign scores of the individual cats receiving the FCP 3Avaccine were compared to those of the ten control cats, a significantdifference (p<0.5) was observed on days 15, 18, 19, 20, 23, 24, 25, 26,27 and 28 following challenge. Both the intramuscular route and thesubcutaneous route of administering the FCP 3A vaccine were foundeffective.

[0114] Similarly, when the daily fever/clinical sign scores of the FCP3B vaccine group were compared with those of the controls, a significantdifference (p<0.05) was obtained on days 6, 13, 16, 17, 18, 19, 20, 23,24, 26 and 27 following challenge.

[0115] In conclusion, these results demonstrate the efficacy of the FCPfraction in the FCP 3A vaccine and its 1:5 fractional dose FCP 3B inprotecting vaccinated cats against virulent FCP challenge.

1. A vaccine composition against feline Chlamydia psittaci infectionscomprising an immunogenically active component consisting of outermembrane antigens derived from inactivated feline Chlamydia psittaci ina dose of at least 50-1,000 ug/dose, an adjuvant, and a veterinarypharmaceutically acceptable carrier or diluent therefor, wherein saidvaccine composition is purified of toxic immunogenic egg yolk sacsubstances, said vaccine composition prepared by the process comprising:(a) culturing the feline Chlamydia psittaci in egg yolk sac; (b)subculturing said egg yolk sac culture containing the feline Chlamydiapsittaci in a suitable cell line to remove toxic immunogenic substancespresent in the egg yolk sac culture; (c) harvesting the feline Chlamydiapsittaci from the contents of the subculture, said contents selectedfrom the group consisting of infected cell line cells, subculturesupernatant and a combination thereof; (d) inactivating the felineChlamydia psittaci with inactivating agents; (e) extracting outermembrane antigens from the inactivated feline Chlamydia psittaci; and(f) mixing the inactivated feline Chlamydia psittaci with the adjuvantand physiologically acceptable carrier or diluent.
 2. A vaccinecomposition of claim 1, wherein said feline Chlamydia psittaci areisolated from feline specimens selected from the group consisting ofvaginal, cervical, nasal, ocular, pneumonic, gastral fluids, secretions,discharges or scrapings, and combinations of any of the foregoing. 3.The vaccine of claim 1, wherein the cell line subculture is harvested upto six times comprising the steps of: (i) adding new chlamydiapropagation media to the cell line subculture following the firstharvest; (ii) incubating the cell line subculture; (iii) harvesting thefeline Chlamydia psittaci from the contents of the subculture, saidcontents selected from the group consisting of infected cell line cells,subculture supernatant and a combination thereof; and (iv) repeatingsteps (i)-(iii) up to five times.
 4. A vaccine composition of claim 1,wherein said adjuvant is selected from the group consisting ofsurfactants, polyanions, polycations, peptides, tuftsin, mineral oilemulsions, immunomodulators, ethylene/maleic anhydride copolymer,copolymer of styrene with a mixture of acrylic acid and methylacrylicacid and combinations of any of the foregoing.
 5. A vaccine compositionof claim 4, wherein said adjuvant is one of ethylene/maleic anhydridecopolymer, copolymer of styrene with a mixture of acrylic acid andmethylacrylic acid, mineral oil emulsion or a combination of any of theforegoing.
 6. A vaccine composition of claim 1, wherein said adjuvant isone of saponin and aluminum phosphate or a combination thereof.
 7. Avaccine composition of claim 1, wherein said adjuvant comprises from0.01 to 50% w/v or v/v.
 8. A vaccine composition of claim 1, whereinsaid veterinary pharmaceutically acceptable carrier or diluent comprisessaline or Eagle's minimum essential media.
 9. A vaccine composition ofclaim 1, wherein said immunogenically active component is incorporatedinto liposomes.
 10. A vaccine composition of claim 1, wherein saidimmunogenically active component is incorporated into sustained releasepolymer(s).
 11. A vaccine composition of claim 1, wherein saidimmunogenically active component is conjugated to a biological compoundselected from the group consisting of polysaccharides, peptides,proteins, and combinations of any of the foregoing.
 12. A vaccinecomposition of claim 1, in a dosage unit form derived from at least2×10⁴ inactivated feline Chlamydia psittaci.
 13. A vaccine compositionof claim 1, in parenteral injectable form.
 14. A method of preventingfeline Chlamydia psittaci infection in felines comprising administeringto a feline in need of such prevention of effective amount of thevaccine composition of claim
 1. 15. A method of claim 14, wherein thevaccine composition is parenterally administered.
 16. A method of claim15, wherein said parenteral administration is carried outsubcutaneously.
 17. A method of claim 15, wherein said parenteraladministration is carried out intramuscularly.
 18. A method according toclaim 14, wherein said vaccine composition is administered at least twotimes, each administration separated by about 14 to about 30 days.
 19. Amulti-vaccine composition comprising the vaccine composition of claim 1,and at least one vaccine composition directed against a pathogenselected from the group consisting of feline leukemia, panleukopenia,calici, rhinotracheitis, feline acquired immunodeficiency disease,rabies, feline infectious peritonitis, toxoplasmosis, and Borreliaburgdorferi, and combinations of any of the foregoing.
 20. A vaccinecomposition comprising an immunogenically active component consisting ofouter membrane antigens derived from inactivated feline Chlamydiapsittaci in a dose of at least 50-1,000 ug/dose, an adjuvant and aveterinary pharmaceutically acceptable carrier or diluent therefor,wherein said vaccine composition is purified of toxic immunogenic yolksac substances.
 21. The vaccine of claim 1, wherein said subculturingcomprises one passage in a suitable cell line.
 22. The vaccine of claim1, wherein said suitable cell line comprises dog kidney cells.
 23. Avaccine composition of claim 23, wherein the inactivating agents areselected from the group consisting of binary ethylenimine,beta-propriolactone, formalin, merthiolate consisting of thimerosal andsodium ethyl-mercurithio-salicylate, thimerosal, glutaraldehyde, sodiumdodecyl sulfate, an alkylaryl polyether alcohol surfactant, andcombinations thereof.
 24. A vaccine composition of claim 23, whereinsaid inactivating agent is thimerosal.